1. Technical Field
The present invention relates to a novel sphingolipid ceramide N-deacylase (hereinafter often referred to as xe2x80x9cSCDasexe2x80x9d) having a wide substrate specificity. Furthermore, the present invention relates to methods for enzymatically producing lysosphingolipid or sphingolipid derivatives using the SCDase which is useful in the fields of medicine, carbohydrate engineering, cell engineering, and the like, and the lysosphingolipid or sphingolipid derivatives obtained by such a production method. Moreover, the present invention relates to a gene which encodes a polypeptide having an SCDase activity useful in sphingolipid technology. Also, the present invention relates to a method for industrially producing a polypeptide having an SCDase activity and a recombinant polypeptide thereof using a transformant to which the gene is introduced, a probe or primer which hybridizes to the gene, and an antibody or a fragment thereof which specifically binds to the polypeptide.
2. Description of the Background Art
In recent years, attention has been paid to various physiological functions of a sphingolipid as well as a glycerolipid as components constituting the cell membrane lipid of eucaryotes. An SCDase which acts on the sphingolipid to form a fatty acid and a lysosphingolipid is not only useful for the elucidation of the physiological actions of the lyso-form sphingolipid but also considerably important in the field of sphingolipid technology such as preparation of sphingolipid derivatives and labeling of a sphingolipid.
xe2x80x9cSphingolipidxe2x80x9d is a generic name for lipids having a long-chain base sphingoid such as glycosphingolipids, sphingophospholipids (involving sphingophosphonolipids) and ceramides. Sphingolipids, which have a ceramide having a long-chain fatty acid with a nonuniform chain length bonded via an acid-amide bond to the amino group of the sphingoid as the common structure, are widely distributed in lower animals to higher animals. Recently, it has been clarified that these sphingolipids participate in important roles in biological activities of cell proliferation, induction of differentiation, apoptosis and the like. Also, attempts have been made to employ these sphingolipids in cosmetics and the like since they are constituents of the cell surface layer.
On the other hand, N-deacylated sphingolipids, in which the fatty acid bonded via an acid-amide bond to the amino group of the sphingoid in the sphingolipid has been eliminated, are called lysosphingolipids. It has been clarified that these lysosphingolipids have biological activities similar to those of the sphingolipids.
Moreover, when re-acylated lysosphingolipids having a free amino group in the sphingoid moiety are useful as starting materials for synthesizing lysosphingolipid derivatives (sphingolipid derivatives). For example, sphingolipids having a uniform fatty acid composition or those differing in the fatty acid chain length from the starting ones can be re-synthesized thereby. It is also possible to obtain sphingolipids labeled with chromophores, radioisotopes such as 14C and the like. Furthermore, lysosphingolipids can be immobilized onto a carrier using the free amino group thereof.
In general, naturally occurring glycolipids such as glycosphingolipids show wide molecular variety depending on a fatty acid moiety even if they have the same carbohydrate chain. For example, Forssman glycolipid (Gb5Cer) derived from a horse kidney includes at least ten different molecular species depending on its fatty acid moiety. Recently, it has revealed that the existing form or antigenicity of glycolipids in a lipid bilayer are greatly influenced by their fatty acid molecular species. Thus, attention has been paid to the structure of a fatty acid in view of the physiological function of a glycolipid. It has been further found that the substrate specificity of enzymes relating to the degradation and synthesis of sphingolipids (including glycolipids and sphingomyelin) depends on fatty acid molecular species.
In order to elucidate the above subject, it has been required to develop technique for simply and easily converting naturally occurring sphingolipids including heterologous fatty acid molecular species into glycolipids having a single fatty acid species. It has also been desired to prepare a fluorescence-labeled sphingolipid by substituting a fatty acid of a sphingolipid with a fluorescent substance since such a fluorescence-labeled sphingolipid is expected to be not only a reagent important for contributing to the elucidation of intracellular metabolism or a transport route of sphingolipids but also a highly sensitive substrate for enzymes synthesizing or degrading a sphingolipid.
Known methods for producing lysosphingolipids include chemical methods, enzymatic methods and microbial methods.
The chemical methods include hydrazinolysis and alkaline hydrolysis in an alcohol solvent. When a glycosphingolipid containing sialic acid (i.e., ganglioside) is treated by these methods, the deacylation of the sialic acid moiety also proceeds at the same time. In the case of a glycosphingolipid containing aminosugar, the N-acetyl group is liberated and thus a de-N-acetyl lysoglycolipid is formed. It is therefore necessary that, after the completion of the deacylation, a protecting group is selectively introduced into the amino group in the lipid moiety and the sialic acid moiety is re-acylated followed by deprotection. Various by-products are formed by these procedures. That is to say, the production of lysoglycolipids by these chemical methods require great labor and technical skill. In addition, it is very difficult to prepare a lyso-form of a polysialoganglioside having plural sialic acids, such as GQ1b, in accordance with the conventional chemical methods.
On the other hand, there have been known chemical methods for obtaining the lyso-form of a sphingomyelin which is a sphingophospholipid. A generally known example of the chemical methods is one comprising hydrolysis with hydrochloric acid in an alcohol solvent. According to this method, however, not only natural a D-erythro (2S,3R) stereoisomer but also an L-threo (2S,3S) stereoisomer are formed, which reduces the yield of the final product and it is very difficult to separate these isomers from each other. When sphingomyelin is treated by the known methods, a choline phosphate group might be possibly liberated, which reduces the yield of the final product.
On the other hand, there have been known methods wherein enzymes forming lyso-forms from glycosphingolipids are employed. However, the method using a ganglioside ceramidase produced by an actinomycetes of the genus Nocardia fails to provide any neutral glycolipid of the lyso-form due to the substrate specificity of the enzyme (JP-A-64-60379 (the term xe2x80x9cJP-Axe2x80x9d as used herein means an unexamined published Japanese patent application). The method using an enzyme produced by an actinomycetes of the genus Rhodococcus or processed cells thereof fails to provide the lyso-form of any acidic glycolipid (ganglioside) (JP-A-6-78782).
An enzyme capable of hydrolyzing a bond between a sphingosine base and a fatty acid of a ceramide, which is called ceramidase (EC 3.5.1.23) [Journal of Biological Chemistry, 241:3731-3737 (1966); Biochemistry, 8:1692-1698 (1969); Biochemica et Biophysica Acta, 176:339-347 (1969); and Science, 178:1100-1102 (1972)], cannot hydrolyze a bond between a sphingosine base and a fatty acid in the ceramide moiety of a glycolipid. Namely, none of known enzymes can widely act on sphingolipids involving glycosphingolipids (ganglioside, neutral glycolipids) and sphingomyelins.
With regard to use of a microorganism or its extract, an actinomycetes of the genus Streptomyces capable of producing a glycosphingolipid ceramide deacylase is employed in a method described in JP-A-7-107988. In this method, a glycosphingolipid is added to the medium and converted into the lyso-form therein. However, this method is also poor in efficiency. Owing to the substrate specificity, moreover, the enzyme cannot act on ganglioside GM3 and neutral glycolipids (i.e., lactosyl ceramide, glycosyl ceramide and galactosyl ceramide). Thus it is impossible to obtain the lyso-forms of these glycolipids by this method.
As discussed above, the conventional chemical, enzymatic and microbial methods for producing lysosphingolipids suffer from such troubles that undesired by-products are formed, great labor and technical skill are required, or the substrate is restricted. Furthermore, these methods can achieve only poor efficiency.
As a common structure, sphingolipids have a ceramide structure in which a long-chain fatty acid having a nonuniform chain length bonded to the amino group of the sphingoid via an acid-amide bond. With regard to the method for producing sphingolipids or sphingolipid derivatives by modifying or substituting the long-chain fatty acid of sphingolipids, methods are known in which they are synthesized chemically or enzymatically using a lysosphingolipid as the starting material which lacks the fatty acid bonded by the acid-amide bond to the amino group of the sphingoid in the sphingolipid.
As the chemical method, there are methods in which a fatty acid or a fatty acid derivative is condensed to the lyso-form amino group by the following techniques. For example, known are a method in which a fatty acid active ester (for example, N-hydroxysuccinimide ester of a fatty acid) is used, a method in which a fatty acid and a coupling agent (for example, carbonyldiimidazole, dicyclohexylcarbodiimide or the like) are used, a method in which a fatty acid anhydride is used, a method in which a fatty acid chloride is used, and the like.
Methods in which a lysoganglioside is used as the lyso-form of an acidic glycolipid are reported in Methods in Enzymology, 138:319-341 (1987), European Patent 373039 B1 (1994) and European Patent 765883 A1 (1997). Also, a method in which a sphingosyl-phosphorylcholine (lysosphingomyelin) is used as the lyso-form of sphingophospholipid is described in Journal of Lipid Research, 28:710-718 (1987).
According to these methods, side reactions (for example, O-acylation and the like) occur in some cases, so that it is necessary to employ complex steps for use of protecting groups, purification and the like, in order to obtain an N-acylated product selectively. Also, when it is required to acylate only the amino group of the sphingoid in a sphingolipid selectively which has an amino group other than the amino group of the sphingoid, such as de-N-acetyllysoganglioside which is obtained by chemically deacylating a ceramide ciliatine which is one of sphingophosphonolipids or a glycosphingolipid containing aminosugar, complex steps, such as a step of introduction of protecting groups, partial acylation, partial deacylation after the acylation, and a step of selective N-acylation after incorporation of de-N-acetyllysoganglioside into liposomes, are required, and therefore it is difficult to conduct the selective acylation.
On the other hand, an enzymatic synthesis method is described in International Publication No. WO 94/26919. In this method, a condensation reaction is carried out by lipase in an organic solvent, so that a substantially water-free organic solvent is required and the substrate is limited depending on its solubility. International Publication No. WO 94/26919 discloses an enzymatic synthesis method of a ceramide and a hybrid ceramide, but the reaction is not specific so that the formation of O-acylated products is found. Furthermore, when the substrate has a plurality of amino groups similar to the case of the chemical synthesis methods, it is difficult to carry out the specific reaction with only the amino group of the sphingoid.
As described above, in the previous method for synthesizing sphingolipids or sphingolipid derivatives by chemically or enzymatically modifying or substituting the long-chain fatty acid in the sphingolipid, undesirable by-products are formed, and the substrate is limited. Additionally, in the previous methods, the lysosphingolipid which lacks a fatty acid bonded by an acid-amide bond to the 2-position of the sphingoid in the sphingolipid is used as the starting material. Therefore, when intended sphingolipids or sphingolipid derivatives are synthesized, it is required to prepare a lysosphingolipid prior to the synthesis.
Furthermore, when the above-described SCDase useful in the field of sphingolipid technology is produced from an enzyme producing organism industrially advantageously, the amount of the naturally existing enzyme is small or, in order to induce the production of the enzyme, it is necessary to culture the enzyme producing organism by adding a ganglioside mixture to the medium, so that free fatty acids are formed in the culture medium and enzymes other than the SCDase, such as a sphingomyelinase and the like, are simultaneously produced, thus causing a difficulty in separating and purifying the SCDase of interest from these free fatty acids and the enzymes contaminated.
Consequently, great concern has been directed toward the development of a method by which this enzyme can be produced with a more lower cost and higher purity.
Although there are reports on the purification of the SCDase from various enzyme producing organisms as described above, there are no reports on the amino acid sequence and the structure of the SCDase, so that the amino acid sequence and gene structure are entirely unclear and therefore, it is difficult to produce an SCDase by means of genetic engineering techniques.
An object of the present invention is to provide an SCDase having a wider substrate specificity than those of the conventionally known glycolipid ceramide deacylases.
Furthermore, an object of the present invention is to provide a method for enzymatically producing a lysosphingolipid, which is useful in the field of sphingolipid engineering, using the above SCDase in an industrial scale without producing by-products.
Moreover, an object of the present invention is to provide a microorganism capable of producing an SCDase for use in the above-described method.
Still furthermore, an object of the present invention is to provide lysosphingolipids and lysosphingolipid derivatives obtained by the above-described method.
Still moreover, an object of the present invention is to provide a production method for specifically synthesizing sphingolipids or sphingolipid derivatives having a modified or substituted long-chain fatty acid bonded to the sphingoid from not only a lysosphingolipid but also a sphingolipid.
Also, an object of the present invention is to provide sphingolipids or sphingolipid derivatives obtained by the production method.
Additionally, an object of the present invention is to provide a gene which encodes a polypeptide having an SCDase activity; a method for industrially producing a polypeptide having an SCDase activity using a transformant introduced to the gene, and such a recombinant peptide; a probe or primer which hybridizes to the gene; and an antibody or a fragment thereof which specifically binds to the polypeptide.
The present invention mainly relates to the following 1) to 19):
1) an SCDase having physicochemical properties of:
(i) acting on a ceramide moiety in the molecule of a sphingolipid and forming a lysosphingolipid and a fatty acid;
(ii) acting on neutral glycosphingolipids, acidic glycosphingolipids, sphingomyelins and ceramides;
(iii) having an optimum pH value range of from 5 to 8.5; and
(iv) having an optimum temperature of about 40xc2x0 C.;
2) a method for producing an SCDase which comprises:
culturing a strain belonging to the genus Pseudomonas or Shewanella capable of producing an SCDase to produce an SCDase; and
recovering the SCDase from the culture;
3) a method for producing a lysosphingolipid which comprises:
(A) treating a sphingolipid with the above SCDase to obtain a reaction mixture; and
recovering a lysosphingolipid from the reaction mixture, or
(B) a method for producing a lysosphingolipid which comprises:
subjecting a sphingolipid to a contact reaction with a microorganism capable of producing an SCDase to obtain a reaction mixture; and
recovering a lysosphingolipid from the reaction mixture;
4) a lysosphingolipid which is obtained by the above method 3);
5) a method for producing a lysosphingolipid derivative which comprises subjecting the lysosphingolipid of above 4) to a substitution reaction;
6) a lysosphingolipid derivative which is obtained by the above method 5);
7) a method for producing a sphingolipid or a sphingolipid derivative, which comprises enzymatically reacting a sphingolipid with an aliphatic carboxylic acid having or free of a marker using an enzyme which can specifically hydrolyze an acid-amide bond between a sphingoid and a fatty acid in a sphingolipid to obtain another sphingolipid or sphingolipid derivative having a different fatty acid chain;
8) a method for producing a sphingolipid or a sphingolipid derivative, which comprises enzymatically reacting a lysosphingolipid with an aliphatic carboxylic acid having or free of a marker using an enzyme which can specifically hydrolyze an acid-amide bond between a sphingoid and a fatty acid in a sphingolipid to obtain a sphingolipid or sphingolipid derivative;
9) a method for producing a sphingolipid or a sphingolipid derivative, which comprises enzymatically reacting at least two different sphingolipids using an enzyme which can specifically hydrolyze an acid-amide bond between a sphingoid and a fatty acid in a sphingolipid to obtain other sphingolipid or sphingolipid derivative having an exchanged fatty acid chain;
10) the method for producing a sphingolipid or a sphingolipid derivative, which comprises using a microorganism which is capable of producing the enzyme, instead of the enzyme in the above 7) to 9);
11) a sphingolipid or sphingolipid derivative, which is obtained by any one of the above methods 7) to 10);
12) a bacterium belonging to the genus Pseudomonas or Shewanella which is capable of producing an SCDase, or a mutant thereof;
13) an isolated gene which encodes a polypeptide having an SCDase activity.
14) a recombinant vector which comprises the gene of the above 13);
15) a transformant to which the recombinant vector of the above 13) is introduced;
16) a method for producing a polypeptide having an SCDase activity, which comprises:
culturing the transformant of the above 15) to produce a polypeptide having an SCDase activity; and
recovering the polypeptide from the culture;
17) a recombinant polypeptide having an SCDase activity encoded by the gene of the above 13), which is obtained by culturing the transformant of the above 15) to produce a recombinant polypeptide, and recovering the recombinant polypeptide from the culture;
18) a synthesized oligonucleotide probe or primer which specifically hybridizes to the gene of the above 13); and
19) an antibody or a fragment thereof obtained by using the polypeptide of the above 17) or a portion thereof, which specifically binds to the polypeptide of the above 17).